Proteins must fold correctly into a defined three-dimensional structure in order to properly function. Several human diseases, includingCreutzfeldt-Jacob disease and cystic fibrosis, can be considered protein folding diseases because proteins fail to achieve active conformations in their proper cellular compartment. Within the crowded cellular milieu, newly translated proteins are prone to aggregation and nonproductive premature folding because of the exposure to the aqueous environment of hydrophobic residues which are normally sequestered within the folded protein. Molecular chaperones are excellent candidates for proteins which bind to and protect nascent chains from problematic interactions because of the nature of theirtransient interactions with hydrophobic polypeptide segments. Data collected in a variety of systems, in vivo and in vitro, indicate a pathway(s) of protein folding from the time proteins are nascent chains on ribosomes until they are either properly folded or degraded. Molecular chaperones play important roles in these processes. The focus of experiments described in this proposal is to determine the function of Hsp70 (heat shock protein 70K) and Hsp40 as molecular chaperones chaperone using the budding yeast Saccharomyces cerevisiae as a model system. More specifically, the focus is on the role of molecular chaperones during the early events of protein folding on the ribosome. The cytosol of S. cerevisiae contains two classes of abundant, functionally distinct Hsp70s (SSA and SSB proteins) and three Hsp40s (Sisl, Zuol and Ydjl). Of these chaperones, Ssb, Sisl and Zuol associate with translating ribosomes, suggesting a role early in the life of a protein. Using a combination of genetic and biochemical approaches we will analyze the function of Ssb, Zuol and Sisl in translation and early steps of protein folding. Hsp70s and Hsp40s are known to function cooperatively as chaperones. However, the functional relationship between them is not well understood. Our goal is to understand the functional relationship between the two Hsp40s (Sisl and Zuol) and one Hsp70 (Ssb) which are associated with the ribosome. More specifically, we will ascertain through genetic and biochemical experiments whether these two DnaJs functionally and physically interact with Ssb. In addition, we will determine the sequence of interactions of a nascent chain with the ribosome-associated chaperones.